The vascular myogenic response has been recognized for nearly 100 years and is likely to underlie the physiological mechanisms responsible for the generation of basal vascular tone and for the regulation of blood flow. Although a number of hypotheses have evolved to explain the vascular myogenic response to stretch, there is little information available as to the intracellular mechanistic events that couple the physical stimulus to the contractile apparatus. The overall goal of this proposal is to investigate several important hypotheses concerning the intracellular signalling pathways thought to be involved in the myogenic response. These include: 1) that calcium is a major transducing signal for initiating the myogenic response; 2) that the sensitivity of the contractile apparatus to Ca2+ is modulated by pressure or stretch; and 3) that protein kinase C is involved in mediating sustained myogenic contraction. To address these hypotheses the proposed studies will include measurement of the effect of pressure/stretch on intracellular Ca2+ in isolated arterioles using fura-2 ratiometric fluorescence microscopy. this will consist of accurate temporal characterization of the relationship between the pressure change, Ca2+, and the arteriolar diameter response. Permeabilized arterioles will be used to establish the relationship between Ca2+ and contraction and to determine whether pressure or protein kinase C can alter the relationship. Biochemical determinations of protein kinase C activity during increases in pressure will be used to directly establish the relationship between activation of this protein kinase and the myogenic response. In addition, fluorescence immunocytochemistry will be used to identify protein kinase C in isolated vascular smooth muscle cells before and after cell stretch. Distributional changes in protein kinase C will be analyzed from 3D images acquired with a digital imaging microscope. Preliminary data suggest that both Ca2+ and a change in the calcium sensitivity of the contractile machinery play a role in the myogenic response. A novel hypothesis arising from these preliminary findings is that the temporal dominance of these two mechanisms during the myogenic response differs such that initial myogenic activation is primarily Ca2+ dependent whereas sustained myogenic constriction involves altered sensitivity of the contractile apparatus which may involve protein kinase C. The information provided by these studies will not only be useful in increasing our basic understanding of the origin of vascular smooth muscle tone but also in developing new insights into vascular physiology. These insights may assist in developing new therapeutic approaches to pathological states of altered vascular tone such as hypertension.